JPH083488B2 - Concentrated immunochemistry test strip - Google Patents

Abstract

A method and device for determining the presence of an analyte in a sample suspected of containing the analyte is disclosed. The method involves contacting a test solution containing the sample and a first member of a specific binding pair with an end portion of a strip of bibulous material capable of being traversed by the test solution through capillary action. The first member of a specific binding pair is capable of binding the analyte. The strip contains a second member of a specific binding pair integral therewith for concentrating and non-diffusively binding the first sbp member at a small situs on the strip separated from the end portion of the strip. The detectible signal is produced in relation to the presence of the analyte in the test solution. The test solution passes through the situs as the test solution traverses the bibulous material. After the test solution has been allowed to traverse at least a portion of the strip, the strip is contacted with a developer solution containing members of a signal producing system in a manner that provides contact of the developer solution with the small situs following its contact with the test solution. The strip is then contacted with any remaining members of the signal producing system. The detectible signal produced at the situs is then compared with the signal detectible at a portion of the strip other than the situs to determine the analyte in the sample. In one embodiment of the invention the signal produced at the small situs has a sharp-edged distinctive pattern that provides a sharp contrast to the signal produced at adjacent sites on the strip when analyte is present in the test solution.

Description

Description: BACKGROUND OF THE INVENTION Field of the Invention It is a flower of immunoassay research that it is possible to use the natural receptor or antibody corresponding to a particular compound in the assay of that particular compound. Brought. A specific binding pair ("sbp") containing a ligand and a receptor (antiligand) in each assay
A homologous pair of components (usually an immune pair) is used and one of its sbps is labeled with a label that produces a detectable signal. Performing the immunoassay results in a distribution of signal label between the signal label bound to the complex of sbp members and the unbound signal label. Discrimination between bound and unbound signal label can be achieved by physically separating the bound signal label from the unbound signal label or by modulating the detectable signal of the bound and unbound signal label.

Immunoassays are commonly subjected to quantitation of a wide variety of compounds of interest in clinical laboratories and require relatively sophisticated equipment and discreet techniques. Immunoassays are aimed at semi-quantitative or qualitative results and have not been widely put to practical use when they are non-experimental technicians for measurement (for example, at home or in a hospital). A simple and rapid screening test that can be performed in the clinical laboratory as well as by non-experts will save a lot of money.

There are many things to consider about the advances in immunoassays. One is the substantial discrimination between the signal generated from the bound signal label and the signal generated from the unbound signal. Minimization of interference by endogenous substances in the sample that may contain the measured compound must also be considered. Also, consideration should be given to detecting the signal and facilitating measurement of the concentration within the concentration range of interest. Other factors include ease of reagent preparation, accuracy required for sample and reagent solution preparation and measurement, reagent storage stability, number of protocol operations, and technique and accuracy required for each operation. That is, in developing assays that can be performed by unskilled personnel (eg, assays that can be performed at home, in forensics, or by practitioners), minimize the variability of results obtained due to differences in protocol implementation. Or,
Or the method of performing the various operations should be simple.

2. Description of the Related Art A test device for measuring sample properties, in particular for measuring substances in liquid samples, is disclosed in US Pat. No. 4,094,647. An apparatus for thin layer chromatography and a chromatographic test method are disclosed in US Pat. No. 4,384,958. Immunoassays that drive labeled antibodies out of immobilized analyte analogs are disclosed in US Pat. No. 4,434,236. A myoglobin detection device and method is disclosed in US Pat. No. 4,189,304. Test strips for analyzing substances dissolved in liquids are disclosed in US Pat.
4,438,067. A multi-layer test device and method for quantifying liquid sample components is described in U.S. Pat. No. 4,160,00.
It is described in No. 8. An antigen measurement method using a labeled antigen using an insoluble antibody is disclosed in JP-A-48-5925 (1980).
25).

Concentration zone method in heterogeneous immunoassay
It is disclosed in US Pat. No. 4,366,241. U.S. Patent 4,1
68,146 describes a test strip immunoassay. U.S. Pat.Nos. 3,990,850 and 4,055,394
No. describes the diagnostic test card. A method for automated quantitative analysis of biological fluids is described in US Pat. No. 4,327,073. The chromogenic support immunoassay is described in International Application No. PC.
It is disclosed in T / US83 / 01887.

Many patents and patent applications provide extensive literature on a variety of techniques for producing detectable signals in immunoassays. The following list is
It is merely exemplary of some such techniques that are relevant to the present invention. The following is a list of US patents and patent applications and types of signs.

U.S. Pat. Nos. 3,646,346, Radioactive Labels; 3,654,090, 3,791,
932 and 3,817,838, Enzyme Label (En
No. 3,996,345, Fluorescer-Quencher Label
s); No. 4,062,733; Radioactive Label; No. 4,067,959; Fluorescer or Enzyme Label; No. 4,104,029; Chemiluminescent Label; and No. 4,160,645, Non. -Non-Enzymatic Catalyst Label. U.S. Pat. No. 3,966,879 describes electrophoresis using antibody zones and 4,120,945 describes RIA in which a labeled analyte is first attached to a solid support via an antibody. U.S. Pat.No. 4,233,402, enzyme pair label, US Pat.
No. 0,450 uses a chemically induced fluorescent label and No. 4,287,300 uses an anionic enzyme label.

SUMMARY OF THE INVENTION The method and apparatus of the present invention comprises an analyte.
It is useful for determining the presence or absence of analytes in samples suspected of containing. The device of the present invention is a strip of bibulous material through which the test solution can pass by capillary movement. The test solution comprises the sample and the first component of the specific pair ("sbp component") capable of binding the analyte. The strips integrally include a second sbp component for non-diffusion binding of the first sbp component at a small site away from the end of the strip that contacts the test solution. Typically, the second sbp component binds to the complex created by the binding of the analyte and the first sbp component. The detectable signal is generated by a signal generating system. That is, after the test solution has been permeated into the absorbent strip, the strip is then applied to a suitable component of the signal generating system used, such as that described below (page 28, line 16 to 36).
(From page 3, line 3, and page 56, line 13 to page 57, line 19)
As such, it is contacted with other components conventional in the art, such as labels or enzyme-catalyzed or non-enzyme-catalyzed, which conjugates with the sbp component to form a signal-generating system. In this way, a signal is generated according to the amount of the analyte in the test solution. In one aspect, the analog of the analyte is
Non-diffusively bonded to the strip, at least between the portion of the strip in contact with the test solution and the said small area.

In the method of the invention, contacting the end of the strip away from the small area with the test solution causes the test solution to penetrate the absorbent material by capillary migration. The strip is contacted with a developer solution containing the signal-generating components, and then the remaining signal-generating components (neither in the test solution nor in the developer solution, nor are they originally present in the strip). If there is a component that does not exist, contact it. At least a portion of the test solution contacts the subsite before the developing solution contacts the subsite. The detectable signal present in the small site is then compared to the detectable signal in the portion of the strip other than the small site to quantify the analyte in the test solution.

In a particular embodiment of the present invention, when analyte is present in the test solution, the signal generated at the small site is a sharp and distinct pattern that is in sharp contrast to the signal generated at the portion of the strip other than said small site. Have.

In another aspect of the invention, the second sbp component is non-diffusively bound to the one subsite of the strip via the particles non-diffusively bound to the one subsite. If the second sbp component can bind to the first sbp component even if the first sbp component does not bind to the analyte, an analog of the analyte that can bind to the first sbp component has the small site and the end. Non-diffusively coupled to the strip between.

The method and apparatus of the present invention will be described later (from page 57, last line to 59).
It is particularly suitable for the quantification of multiple analytes in a test solution, such as page 3 to line 3, and page 61 to line 16 to page 62 to line 12. The presence of one or more analytes in the test solution can be easily determined in a single strip. Furthermore, the method of the present invention allows analytes such as drugs to be detected without the need for reference material or instrument operation. In addition, the device of the present invention, as described later (page 62, line 13 to page 63, line 7), is used in other conventional signal generating systems required to generate a detectable signal in the presence of an analyte. Ingredients, eg below (page 28)
(From line 16 to page 36 line 3), such as an isotope or an enzyme-catalyzed or non-enzymatic catalyst, combined with an appropriate label that forms a signal-generating system by conjugating with the sbp component,
Further, if necessary, it can be provided as an analyte measuring kit which is packaged in combination with an auxiliary reagent for signal generation.

Embodiments As mentioned above, the present invention relates to a method and an apparatus for quantifying an analyte in a sample suspected of containing the analyte. A test solution is prepared by placing the sample, the first sbp component capable of binding the analyte, and the aqueous medium. The ends of the strips of absorbent material that allow the test solution to pass by capillary movement are brought into contact with the test solution. The strip integrally comprises a second sbp component capable of binding to the complex formed from the analyte and the first sbp component. First
The 2sbp component is non-diffusively bound to a small site away from the end of the strip. The test solution penetrates at least a portion of the strip. The strip is then contacted with a developer liquid containing signal generating components. In this method, at least a portion of the test solution contacts the site before the site contacts the developing solution. Then, if necessary,
The strip is contacted with the rest of the components of the signal-generating system (components neither contained in the test solution nor the developer nor present in the strip). The detectable signal at said site is then compared to the detectable signal at the parts of the strip other than that site. A signal is generated at the site depending on the amount of the analyte present in the test solution.

The second sbp component provides a means of concentrating the first sbp component at the site and allowing non-diffusive binding to the strip. First
The 1 sbp component is part of the signal generation system that produces a detectable signal at the site depending on the amount of analyte in the sample. The surface area of said site is essentially smaller than the strip area. The second sbp component is bound directly to the analyte, or directly to the first sbp component (including binding to the signaling label), or by binding to a moiety present only in the complex. It has the property of binding to the first sbp component complex.
If the second sbp component is able to bind directly to the first sbp component and therefore to the uncomplexed first sbp component, an analyte analog that can bind to the uncomplexed first sbp component will bind to the strip. There is. Typically, the analyte analog is non-diffusively bonded to the strip at least between the site and the end.

The means for generating a detectable signal is typically a signal generating system in which one component is conjugated to the sbp component to become the label-sbp component conjugate. The amount of label that produces a detectable signal corresponds to the amount of analyte in the test solution. The signal generating system comprises the label-sbp component conjugate and all other reagents necessary to generate a detectable signal at said site that corresponds to the presence or amount of analyte in the sample.

In a particular embodiment of the invention, the second sbp component is conjugated to a particle that non-diffusively binds to the strip at said site.

The sub-site may be a strip across the direction in which the test solution passes along the strip. The signal generated at the small site may have a distinctive, sharp-edged pattern that is in sharp contrast to the signal generated at other parts of the strip. Typically, the signal generated at the small site is compared to the area of the strip in its vicinity.

The present invention is particularly suitable for the quantification of multiple analytes in a test solution.

Prior to proceeding further with the description of particular embodiments of the invention, a set of terms is defined.

Analyte: The compound or composition to be measured, which is a member of a specific binding pair and may be a ligand, which is typically monovalent or polyvalent antigenic or haptenic, a single compound, or A group of compounds that share at least one common epitope or determinant site or receptor.

Polyvalent ligand analytes are typically poly (amino acids)
(Ie, polypeptides and proteins), polysaccharides,
Nucleic acids, and combinations thereof. Such combinations include bacteria, viruses, chromosomes, genes, mitochondria, nuclei, cell membranes and the like.

The exact nature of the analytes and numerous examples thereof are disclosed in US Pat. No. 4,299,916 (especially columns 16-23), the disclosure of which is incorporated herein by reference.

The analytes of interest include drugs, metabolites,
Includes pesticides and contaminants.

Such drugs include alkaloids. Alkaloids include morphine alkaloids (including morphine, codeine, heroin and dextromethorphan), derivatives and metabolites thereof; cocaine alkaloids (including cocaine and benzoylecgonine), derivatives and metabolites thereof, ergot alkaloids (of lysergic acid). (Including diethylamide); steroid alkaloids; iminazoyl alkaloids; quinazoline alkaloids; isoquinoline alkaloids; quinoline alkaloids (including quinine and quinidine); diterpene alkaloids, their derivatives and metabolites.

Another group of drugs are steroids, which include estrogens, estogens, androgens, andrecorticosteroids, bile acids, cardiac glycosides and aglycones (including digoxin and digoxigenin, saponins and sapogenins), their derivatives and metabolites. . Also included are steroid-like substances such as diethylstilbestrol.

Another group of drugs are 5-6 membered lactams, including barbiturates (eg phenobarbital and secobarbital), diphenylhydantoin, primidone, ethosuximide and its metabolites.

Another group of drugs is aminoalkylbenzenes with alkyl groups having 2-3 carbon atoms, including amphetamines, catecholamines (including ephedrine, L-dopa, epinephrine, narcein, papaverine), and their metabolites.

Another group of drugs are benzene-fused heterocycles, including oxazepam, chlorpromazine, tegretol, imipramine, its derivatives and metabolites, and heterocycles including azepine, diazepine and phenothiazine.

Another group of drugs is the purines, which include theophylline, caffeine, its derivatives and metabolites.

Another group of drugs are marijuana derivatives, including cannabinol and tetrahydrocannabinol.

Another group of drugs are vitamins such as vitamins A, B, including, for example, vitamins B ₁₂ , C, D, E and K, folic acid, thiamine.

Another group of drugs includes prostaglandins with varying degrees and positions of hydroxylation and unsaturation.

The other group of drugs are antibiotics, penicillin, chloromycetin, actinomycetin, tetracycline,
Includes teramycin, its derivatives and metabolites.

Another group of drugs are nucleosides and nucleotides, including ATP, NAD FMN, adenosine, guanosine, thymidine and cytidine, with appropriate sugar and phosphate substituents. Another group of drugs is methadone, meprobamate, serotonin, meperidine, amitriptyline, nortriptyline, lidocaine, procainamide, acetylprocainamide, propranolol, griseofulvin, valproic acid, butyrophenone, antihistamines, anticholinergics (eg atropine).
And various derivatives thereof and metabolites thereof.

Pathology-related metabolites include spermine, galactose, phenylpyruvic acid and porphyrin (I).

Another group of drugs includes aminoglycosides such as gentamicin, kanamycin, tobramycin and amikacin.

Insecticides include polyhalogenated biphenyls, phosphate esters, thiophosphates, carbamates, polyhalogenated sulfenamides, their derivatives and metabolites.

Specific binding pair component ("sbp component"): One or two different molecules that specifically bind to (and thus complement) a particular spatial and polar organization of the other molecule. (Defined as) on the surface or in the cavity. The members of the specific binding pair are called the ligand and the receptor (antiligand). These are usually members of the immunopair, but other specific binding pairs (eg biotin-avidin, hormone-hormone receptors, nucleic acid pairs, etc.) are not immunopairs.

Ligand: is the corresponding receptor naturally present?
Or an organic compound capable of producing a receptor.

Receptor (“antiligand”): A compound or composition capable of recognizing a particular spatial or polar organization of a molecule (eg, an epitopic or determinant site). Examples of receptors include natural receptors such as thyroxine-binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, protein A, complement component C1q.

Ligand or analyte analog: a modified or surrogate ligand capable of competing for a receptor with a similar ligand or analyte
d surrogate) or modified or surrogate analytes, where the modification provides a means of attaching the ligand or analyte analog to other molecules. A ligand analog or analyte analog is usually a ligand or analyte analog at more points (not a requirement) than the replacement of hydrogen by a bond linking the ligand analog or analyte analog to the hub or label. Different from lights. By surrogate ligand or surrogate analyte is meant a compound capable of binding the first sbp component. That is, the surrogate ligand or surrogate analyte can bind the first sbp component in the same manner as the ligand or analyte. A surrogate, on the other hand, is an antibody against the idiotype antibody of the ligand or analyte, for example.

Absorbent material: A porous material with a pore size of at least 0.1 μl, preferably at least 1.0 μ, that facilitates passage of aqueous media by capillarity. Such materials are usually hydrophilic or may be hydrophilic, inorganic powders such as silica, magnesium sulphate and alumina; natural polymeric materials, especially cellulosic materials and materials derived from cellulose, For example, fiber-containing paper (filter paper, cosmatograph paper, etc.); including synthetic or modified natural polymers such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross-linked dextran, agarose, polyacrylate, etc., or Used in combination with other materials such as ceramic materials. The absorbent material can be attached to a support. On the other hand, it is not necessary to supply a support other than the absorbent material itself. The absorbent material is polyfunctional,
Or it can be polyfunctional and allow covalent attachment of the sbp component and attachment of other compounds, forming part of the signaling system.

The binding of the sbp component to the absorbent material is carried out by well known methods commonly described in the literature. For example, "Immobilized Enzymes", Shibata Ichiro, Halstep Press, New York,
1978) and Cattlecassus, Journal of Biological Chemistry (J. Bio. Chem.) 245, p. 3059 (1970).

The absorbent material can be a unitary structure, such as a strip cut sheet, or it can be a particulate material bound to a support or solid surface, such as is used in thin layer chromatography.

If a support is desired or required, the support of absorbent material is typically water-insoluble, non-polar and rigid,
It has the same length and width as the absorbent material, but may be larger or smaller than the absorbent material. A wide variety of synthetic and natural organic or inorganic materials and combinations thereof may be used as long as they do not interfere with the capillary action of the strip or non-specific binding of the assay compound and do not interfere with the signaling system. Examples of polymers include polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly (ethylene terephthalate),
Examples include nylon, poly (vinyl butyrate), glass, ceramics, and metals.

Labeled sbp component: A label conjugated to the sbp component (eg, a catalyst, usually an enzyme) and a member of the signal generating system. The sbp component can bind directly to the analyte, or indirectly to the analyte by binding to the sbp component complementary to the analyte.

Label: The label may be any other molecule or molecule conjugated to an absorbent support, which molecule is optionally selected when two molecules are involved. . In the present invention, the label is a member of the signal generating system, which is conjugated to the sbp component. The label may or may not be an isotope, but is preferably not an isotope. However, isotopes may be preferred to enhance sensitivity when performing radio-autographic measurements using photographic film.

Signal-generating system: A signal-generating system may have one or more components, at least one component being sbp
Label conjugated to the component. The signal-generating system contains all reagents necessary for the generation of a measurable signal. If the first sbp component is not conjugated to the label, the label will typically bind to the sbp component complementary to the first sbp component and will typically be included in the developer. The signal generating system may require other auxiliary components, which are conventional, such as substrates, coenzymes, enhancers, secondary enzymes, activators, cofactors, inhibitors, There are scavengers, metal ions, specific binding substances (necessary for binding signal generating substances), etc., and they are usually used by being included in the developer. The components of the signal generating system may be bound to strips such as coenzymes, substances that react with enzyme products, other enzymes and catalysts. Regarding various techniques for producing a detectable signal in an immunoassay, see (12-13) above.
(Page) As an example, many documents are known and within the scope of conventional techniques. For the convenience of implementing the present invention, the details thereof are as follows. The signal generation system is
It produces a signal which can be detected by conventional external means, usually by measurement of electromagnetic radiation, preferably by visual inspection. For example, typically the signal-generating system comprises a chromogenic substrate and an enzyme, which is enzymatically converted to a dye that absorbs light, phosphorescence or fluorescence in the ultraviolet or visible region.

The signal-generating system may also comprise at least one catalyst (usually at least one enzyme) and at least one substrate as customary auxiliary components, comprising two or more catalysts and a plurality of substrates. You can go out 1
It may include an enzyme composition in which the substrate for one enzyme is the product of the other enzyme. The action of the signal generation system is
The complex formation of the labeled sbp component leads to a product at the small site that produces a detectable signal depending on the amount of catalyst bound to that site.

The signal-generating system produces a compound, usually a signal-generating compound.
In some cases, it may react with other surface-bound compounds, with the associated destruction or destruction. In the signal generation system,
Both enzymatic and non-enzymatic catalysts may be used, but usually at least 1
A seed enzyme catalyst is used. When only one catalyst is used, this catalyst is usually conjugated with the sbp component and
It binds to the site by component complex formation. In addition to the catalyst, there must also be a substrate that undergoes conversion to a detectable signal at the measurable surface. Customary sign sb
The product catalyzed by the p component and obtained by the conversion is a signal generating compound.

Where two catalysts can be used, it may be a combination of enzymatic and non-enzymatic catalysts, and if the product of one catalyst is the substrate of the other catalyst, both are enzymatic catalysts. Good. In this system, only one substrate is required that can be a compound that continuously changes by the catalyst to generate a detectable signal. However, there is usually a series of substrates for the first enzyme and a second compound (a precursor of the compound involved in signal generation, which typically produces a signal generating compound). That is, the product of the first enzyme is reacted with the signal generating compound precursor to supply the signal generating compound.

Usually, hydrolysis or redox reactions are involved.
When performing hydrolysis, derived dye precursors with enzymatically labile bonds and enzymes that catalyze the conversion thereof to insoluble dye products are examples of such systems. When performing a redox reaction, if the second enzyme catalyzes the reaction between the oxidation substrate and the dye precursor, the first enzyme produces the essential oxidation substrate required for the second enzyme.

When using two enzymes, the first enzymatic reaction involves the hydrolysis or redox reaction of the substrate, providing the product which is the substrate for the other enzyme. The first reaction is glucose-6.
-Explained by the catalytic hydrolysis of phosphate to glucose (substrate for glucose oxidase). The second reaction is described by the reaction of oxidizing glucose with glucose oxidase to provide hydrogen peroxide (which enzymatically reacts with the dye precursor to produce a signal generator).

A non-enzymatic catalyst can also be added to the paired catalyst. Enzymes can produce reactants that carry out reactions catalyzed by non-enzymatic catalysts, or non-enzymatic catalysts can produce substrates for enzymes (including coenzymes). A variety of non-enzymatic catalysts that can be used are described in US Pat. No. 4,160,645 (issued Jul. 10, 1979), the relevant parts of which are incorporated herein by reference.

Various combinations of enzymes may be used to provide the signal generating compound. In particular, a combination of hydrolases can be used to generate insoluble signal generators. Also, the combination of hydrolases and oxidoreductases can produce signal generating compounds. In addition, a combination of oxidoreductases can be used to produce insoluble signal generating compounds.

In the enzyme combination, one enzyme is non-diffusively bound to the strip and the other enzyme is conjugated to the sbp component. Furthermore, depending on the type of signaling system or protocol chosen, one or more other components of the signaling system may bind to the strip.

In order to obtain a detectable signal, it is desirable to provide a means to amplify the signal generated by the presence of the label bound to said site. That is, typically the label is preferably a catalyst or a luminescent compound or a radioisotope, more preferably a catalyst. Preferably, the catalyst is an enzyme and coenzyme capable of producing multiple signal generating molecules from a single label.

Enzymes and coenzymes are used that amplify the signal to the desired extent by the production of products that absorb light (eg dyes) or products that emit light upon irradiation (eg fluorophores). It is also possible to guide direct light emission by a catalytic reaction (for example, chemiluminescence). Many enzymes and coenzymes that produce such products are described in US Pat.
No. 4,275,149, columns 19-23 and US Pat. No. 4,318,980, columns 10-14, the disclosures of which are incorporated herein by reference.

In a combination of enzymes of particular interest, there is a relationship in which the product of one enzyme is the substrate for the other enzyme. In such a case, a stable precursor for the labile substrate can be provided and the substrate for the second enzyme can be stored with the first enzyme without starting the reaction prior to use.

A number of catalyst combinations are described in US Pat. No. 4,275,149, columns 23-28, the compositions of which may be used in the present invention. This disclosure is quoted as part of the description.

Of particular interest is the enzyme that produces hydrogen peroxide, and the use of hydrogen peroxide to oxidize dye precursors to dyes. Glycooxidases (eg glucose and galactose oxidase) or heterocyclic oxidases (in combination with enzymes that require hydrogen peroxide to oxidize dye precursors (ie peroxidases such as horseradish peroxidase, lactoperoxidase or microperoxidase)). A combination comprising eg uricase and xanthine oxidase) is preferred. Still other combinations are described in the references. If a single enzyme is used as the label, other enzymes such as hydrolases, transferases and redox enzymes, preferably hydrolases such as alkaline phosphatase and β-galactosidase may be used. Also, luciferases such as firefly luciferase and bacterial luciferase may be used.

Examples of coenzymes used include NAD (H); NADP (H); pyridoxal phosphate; FAD (H); FMN (H) and the like. Usually, coenzymes are used for cycling reactions.
reaction). See U.S. Pat. No. 4,318,980.

The product of the enzymatic reaction is usually a dye or a fluorophore. A number of examples of fluorescent materials are described in US Pat. No. 4,275,149, columns 30-31, which are incorporated herein by reference.

Auxiliary substances: Various auxiliary substances are often used in the assays of the invention. For example, buffers and stabilizers are commonly present in assay systems. Often, in addition to these additives, additional proteins such as albumin, surfactants, especially nonionic surfactants, such as binding enhancers such as polyalkylene glycols, are included.

Minor: A region of a strip of absorbent material, having an area essentially smaller than the strip area. This site may be dot-shaped, linear, curved, strip-shaped, or a pattern formed by dots, lines, curves, strips, or a combination thereof. Typically, the direction in which the test solution penetrates the strip is across this site. Preferably, the signal generated at this site has a sharp and distinctive shape, in sharp contrast to the signal generated at parts other than this small site of the strip. For example, the small site can be marked with the abbreviation or name of the analyte in the test solution, a plus sign, etc. According to the main principle of the invention, this sub-site is remote from the end of the strip in contact with the test solution. In order for the test solution to be well concentrated, the small area must be in contact with the main part of the solution that penetrates the strip.

In the method of the invention, the first s capable of binding an analyte
The bp component is combined with a sample suspected of containing an analyte to form a test solution. The second sbp component, which can bind to the complex formed by the binding of the analyte to the first sbp component, non-diffusively binds to the absorbent strip at the small site. When one end of the strip is contacted with the test solution, the test solution penetrates the strip by capillary action. The amount of the first sbp component bound to the small site via the binding to the second sbp component is
It is related to the amount of analyte in the sample. The signal generating system generates a signal detectable at the site only when the first sbp component is bound, and the signal detectable at the small site is generated at a portion other than the small site of the strip (usually adjacent to the site. The amount of analyte can be quantified by comparison with the signal detectable in (part). The first sbp component binds specifically to the analyte. The second sbp component may be non-diffusively bound at the small site and bound to the first sbp component. Direct binding to the binding site of the 1st sbp component, or the 1st sbp
Indirect binding to the binding site of the analyte binding component may also occur. Binding at a site unique to the analyte-first sbp component complex, which is not present in each component alone, can also occur.

When the first sbp component and the second sbp component are directly bound, the free first sbp component is bound by non-diffusion binding of the analyte analog to the strip at least between said subsite and edge.
It is necessary to remove the components. Typically, if the analyte has a single binding site (eg, it is a drug), or if there is only one sbp component complementary to the analyte, then the second sbp component that binds directly to the first sbp component is selected. . Generally, the amount of analyte analog bound to the strip should be sufficient to bind all the first sbp components in the absence of analyte in the test solution. Usually, an excess of such analog is present.

The test solution moves through the strip by capillarity. The test solution is delivered to the site by capillarity within the strip. Preferably, the strip is contacted with the test solution, followed by capillary migration through the site with the developer. In this case, the developer liquid is brought into contact with the end of the strip with which the test solution is brought into contact. Further, in order to bring the strip into contact with the developer, the end portion of the strip may be brought into contact with the test solution, and then the portion may be immersed in the developer. In any case, before contacting the site with the developer, the first sbp
Concentrate ingredients on the site.

The solvent is usually an aqueous medium in which the concentration of other polar solvents does not exceed about 40% by weight, in particular oxidizing solvents having 1 to 6 carbon atoms, usually 1 to 4 carbon atoms (eg alcohols, ethers etc.). Co-solvents are typically present in amounts not exceeding about 20% by weight.

The pH of the medium is generally 4-11, especially 5-10, preferably about 6-9. The binding affinity of the sbp component is kept moderate,
The pH is selected so that the generation of signal by the signal generating system is optimally maintained. To achieve the desired pH and
Various buffers may be used to maintain pH. Examples of buffers include buffers such as boric acid, phosphoric acid, carbonic acid, Tris, barbital and the like. The buffer used is not limited to a particular buffer, but the preferred buffer may vary from assay to assay.

It is desirable to include about 0.05-0.5% by weight of nonionic surfactant in the sample. Various polyoxyalkylene compounds from 200 to 20,000 Daltons may be used.

Typically, the assay is conducted at a moderate temperature, preferably substantially constant. The temperature at which the assay is carried out and which produces a detectable signal is typically about 4-50 ° C, especially about 10-40 ° C,
More particularly room temperature, ie about 15-25 ° C.

The concentration of analyte in the liquid sample to be assayed will typically vary from about 10 ^-4 to 10 ^-15 M, especially from about 10 ^-6 to 10 ^-14 M. Typically, the concentrations of other reagents are determined, taking into account the concentration and protocol of the analyte of interest.

Most concentrations of different samples and sample solutions in a sample are typically determined by the concentration range of the analyte, but the final concentration of each reagent is typically empirically determined to optimize assay sensitivity over the entire measurement range. decide. In some protocols, each reagent may be used in substantial excess without detrimentally affecting the sensitivity of the assay.

The dimensions of the strip are determined by several factors. If the capillary flow is mainly upward, the length and thickness of the strip regulate the amount of solution that can pass through the site. In order to transfer a large amount of the first solution, the liquid receiving amount of the strip above the site needs to be sufficient to accommodate a desired volume of liquid. Such a volume is not necessary if the strip is to be dispensed mainly into a downward liquid, such as siphoning the test solution. In addition, no such volume is required when the absorbent material is contacted with the strip ends not used for contact with the test solution. It is generally desirable to pass as much of the site as possible to maximize the sensitivity of the assay. However, considering the time required for the assay and the amount of sample used, such requirements are limited. Typically, for upflow strips, the liquid retention volume near the site is greater than about 20 μl, preferably at least 50-20.
It is 0 μl. The downflow strip has a holding capacity of 2
It may be as low as -20 μl, but is preferably 20-200 μl.

The thickness of the strip typically does not exceed 20% of the width, preferably 1-10% of the width, more preferably 2-50% of the width.

The width of the strip is typically relatively narrow (typically no more than 20 mm, preferably no more than 10 mm) in order to protect the reagents and deliver the sample into the limited dimensions. Usually the width of the strip does not exceed about 10 mm, especially about 2-12 mm, preferably about 4-8 mm.

The length of the strip is determined by the concentration of the analyte and the ease of handling and the number of the sites on the strip, and is about 2 to 40 cm, usually about 4 to 25 cm, preferably about 6 to 20 cm. However, any practical length may be used. The structure of the strips is diverse, including fine, medium fine, medium and medium coars.
e), and crude. Generally, the smaller the pore size and the finer the material used, the slower the capillary flow and the better the ability of the sbp component to bind to the strip. The use of coarser porous material results in faster flow rates, but less binding. Material roughness is selected by the binding rate of the sbp component in the particular assay.

The location of the subsite, or the subsite for quantifying multiple analytes, is managed according to the basic principles of the invention. A sufficient amount of test solution is passed by capillary action to the small site and a sufficient amount of analyte is concentrated in the small site to generate a detectable signal relative to the background. That is, it is desirable to place the small site near the end of the strip with which the test solution is contacted (but not close enough to contact a large volume of solution or meniscus). Preferably,
The subportion is at least from such a strip end.
5 mm apart, preferably 8 mm apart. If the test solution can pass through the sub-site by capillary action, the sub-site may be located further from the end. Preferably,
Do not place the subsites more than one-half of the length of the strip from such an end. In this way, the small portion is “separated” from the end. When multiple small sites are provided, the sites may be grouped together in close proximity or dispersed (provided that the signals should not be close enough to be indistinguishable). Therefore, such small portions are usually arranged at a distance of 1 mm or more, preferably at least 3 mm.

Depending on the particular protocol and its role in signal generation, the concentrations of other reagents of the members of the signal generation system may vary. Generally, the concentration of the first sbp component does not exceed 10 ⁴ times the maximum concentration of the analyte when the analyte has a large number of binding sites, and when the monovalent analyte is used, The maximum concentration of 10 ³ should not be exceeded. Typically, the concentration of the first sbp component is about 0.5 times the lowest concentration of analyte to be measured. If the label is not bound directly to the first sbp component, then the reagent that binds to it must be one that binds to the first sbp component,
It must be present in at least an amount equivalent to the lowest concentration of the analyte of interest.

In performing the assay, the protocol typically involves the preparation of a test aqueous solution by mixing a sample, which may contain analyte, with the first sbp component. Samples can be prepared from various test materials such as biological fluids (eg, saliva, blood, serum, plasma, urine, lacrimal fluid, cerebrospinal fluid, etc.), chemical reaction fluids, food waste fluids, and the like.

Strip end (usually the end closest to the site)
Is contacted with the test solution, typically by dipping the ends in the test solution. Generally, wetting of the strip is continued by capillary action at least until the site is wetted. Preferably, half of the strip is moistened with the test solution. When siphoning and penetrating downwards, the entire strip may typically be wetted and excess test solution siphoned from the strip.

Usually, the test solution passes through the strip in a relatively short time. Generally, the passage of the test solution through the strip is
It takes at least 30 seconds to 1 hour, especially about 1 to 30 minutes. The signal usually appears for 30 seconds to 30 minutes, especially about 30 seconds to 5 minutes.

After passing the liquid through at least a portion of the strip, the strip is contacted with a developer liquid having components of the signaling system. This can be done by dipping the strip into the developer, but preferably only the strip ends (previously contacted with the test solution) are contacted with the developer. When the test solution contains the unlabeled first sbp component, the developer has a labeled sbp component capable of binding to the complex formed from the first sbp component and the analyte. Upon contact between the strip edges and the developer, the developer is allowed to penetrate by capillary action into at least the small areas of the strip, preferably until the entire strip is wet.

When using an enzyme as a label, the substrate is usually in substantial excess (higher than Km) so as not to limit the reaction rate. The developer is typically buffered as appropriate for the enzyme system.

After contacting the strip with the developer, the strip is
Contact with the remaining components of the signal-generating system (components not included in the developer or test solution and not present in the strip). Allow sufficient time before measuring the signal to generate the amount of signal generating compound needed to determine the area of the site where the analyte is bound. The generation of a detectable signal indicates whether one or more analytes are present in the sample.

Ligand analytes are characterized by a single binding site (monovalent) or multiple binding sites (multivalent),
Receptor analytes may also have single or multiple binding sites. Polyvalent analytes are typically poly (amino acids), ie polypeptides and proteins, polysaccharides, nucleic acids and combinations thereof. Such combinations include bacteria, viruses, chromosomes, genes, mitochondria, nuclei, cell membranes and the like.

Typically, the multivalent ligand analyte has a molecular weight of at least about 5,000, especially at least about 10,000. In the case of poly (amino acid) s, poly (amino acid) as described above
The molecular weight of is usually about 5,000-5,000,000, especially about 20,000-
1,000,000, and for hormones, the molecular weight is about 5,000
~ 60,000.

Many examples of useful ligands are found in US Pat. No. 4,275,149
No. 12 to 17 columns, which are incorporated herein by reference.

The molecular weight of the monovalent ligand / analyte is usually about 100-
2,000, especially about 125-1,000. The analytes of interest include drugs, hormones, metabolites, pesticides, pollutants and the like.

Many such analytes have been described in U.S. Pat.
No. 9 columns 17-18 are cited here and are hereby incorporated by reference.

In the case of receptor analytes, the molecular weight is usually about
It is 10 ^{4 to} 2 × 10 ⁸ , especially about 3 × 10 ⁴ to 2 × 10 ⁶ . If immunoglobulins (e.g. IgA, IgD, IgE, IgG and IgM), the molecular weight is varied in the range of usually about 160,000～10 ^6. Enzymes typically vary from about 10,000 to 600,000 daltons. Natural receptors typically have a molecular weight of at least about 25,000, may be 10 ⁶ and higher, and include avidin, thyroxine-binding globulin, thyroxine-binding prealbumin, transcortin, membrane surface proteins and the like.

When conjugating a ligand to another molecule or support, the ligand is often modified to provide a particular functional group at a particular site. This modification produces a product called the ligand analog. U.S. Pat.No. 4,275,149
Nos. 18 to 19 describe the ligand analogs in detail, which are incorporated herein by reference.

The strip can be coated with various materials to enhance performance. The coating includes protein coating, polysaccharide coating,
Included are synthetic polymers, sugars and the like, which are used especially to enhance the stability of the material conjugated to the support. These compounds may also be used to improve the binding of substances such as sbp components or signal generating system components to strips.

The strip or the small sites can be activated with reactive functional groups to conjugate organic material to the strip (see US Pat. No. 4,168,146).

The amount of sbp component that binds to the strip at the small site is
Varies depending on the amount required to bind all labeled sbp components. Typically, the amount of sbp component in the small site is at least equal to the amount of analyte passing through the small site and may be 10,000 times or more the amount of analyte.

The second sbp component, the analyte analog, and optionally the components of the signal-generating system can be bound to the strip by adsorption rather than covalent binding, provided that binding must be non-diffusive. This is by contacting the absorbent support with a solution containing a substance that binds to the strip and allowing the strip to dry. Typically, this method is only useful if the absorbent support is relatively hydrophobic or has a high surface charge, and may block proteins, detergents, polysaccharides, or other non-specific binding sites. Post-treatment with substance required.

In a preferred embodiment of the invention, the first sbp component may be non-diffusively bound to particles or beads. The particles or beads can then be applied to the small areas of the strip. Typically, the particles have a means for specifically binding the labeled sbp component or label with less non-specific interactions. The particles or beads are natural or synthetic and have various properties. This material is commercially available and commercially available materials may be modified. Examples of such particles or beads are polystyrene, polyacrylate, polyacrylamide latex particles, or Sepharose, commercially available as Bilgel-p ™.
e. Trademarks), natural materials such as commercially available polysaccharides, especially cross-linked polysaccharides (eg agarose), Sephadex (trademark) dextran, microcrystalline cellulose, starches and the like. Other materials include polyacrylamide, polystyrene, polyvinyl alcohol, copolymers of hydroxyethylmethacrylate and methylmethacrylate, silicones, glass commercially available as Bioglas ™, diatomaceous earth, silica, and the like. The first requirement is that the material does not participate in the signal (usually the absorption of light), i.e. it does not generate a signal different from the rest of the strip at said site prior to contact with the signal generating system.

The particles must be able to bind non-diffusively to the sbp component, which can be covalent or non-covalent.
If the sbp component is covalently attached, the particles should be or capable of being multifunctional. Various functional groups may be present or incorporated. Functional groups include carboxylic acids, aldehydes, amines, amides,
It includes active ethylene such as maleimide, hydroxyl, sulfonic acid, mercaptan and the like.
Methods for linking different compounds to different particles are well known and are well documented in the literature. For example, Journal of Biological Chemistry (J.Biol.Che
m.) 245, p. 3059 (1970).

The length of the linking group varies widely depending on the properties of the compound to be linked, the influence of the distance between the label and the particle on the properties of the label, the crosslinkability of the label, and the like.

The particles should not move to any extent. The size of the particles can vary, but must be such that they penetrate the pores of the absorbent material and become trapped or non-diffusively bound therein. That is, the particle size is typically slightly larger than the minimum pore size of the absorbent material and smaller than the maximum pore size. Usually, the particle size is about 0.1 to 50μ, especially about
0.4 to 10μ, preferably larger than 0.5μ.

Particles with non-diffusive bound sbp components can be used to non-diffusively bind sbp components to strips at tightly defined edge small sites. This can be done in several ways.
Typically, a suspension of particles (often an aqueous suspension) may be applied to the strip. The application may be any standard printing method including the use of electrostatic and laser propeller jets and printing probes or printing. further,
The particles may be supplied with the template. The shape of the site is determined by the cut pattern in which the particles are absorbed by the absorbent strip. Alternatively, the suspension can be transferred to a strip by filling in with a pen or microcapillary tube. When using dry particles,
The particles are delivered by injecting a particle suspension in a gas (typically air) at the desired location. In each case, it is often desirable to depressurize the side opposite the side supplying the particles, especially if the printing method is not used. Vacuum is conveniently applied by placing a sheet of absorbent material on a filter or porous plate that covers the vacuum chamber. The suspension is fed while aspirating air from the material. Regardless of the method of particle delivery, it is usually desirable to wash the site after delivery to remove unbound particles.

The liquid used to suspend the particles is typically aqueous and should not dissolve the particles or compromise or dissociate the bound sbp component. Thickeners and surfactants may be added to limit capillary flow and deliver to tightly restricted sites. Thickeners include polyvinyl alcohol, polypyrrolidone, dextran, glycerol and the like. Surfactants may be ionic (usually anionic) or nonionic.

Various aspects of the invention are described in some detail below. In one aspect, the analyte in the sample is multivalent. The second sbp component, which recognizes the analyte determinant site, is non-diffusively bound to a small site on the strip of absorbent material. Non-diffusion coupling may be performed by any of the above means. A test solution is prepared by mixing the sample suspected of containing the analyte and the labeled sbp component in a volume approximately equal to the liquid capacity of the strip, where the latter
The sbp component binds to the analyte determinant site to form a complex). When the end of the absorbent strip closer to the site is contacted with the test solution, the test solution penetrates the absorbent strip by capillary action. The test solution travels within the strip and passes through the site until the entire strip is wet or the test solution is depleted. Analytes and signs No.
The complex with the 1 sbp component binds to the second sbp component at the site, thereby concentrating the labeled sbp component at the site.
After penetrating the absorbent solution with the test solution, the strip is contacted with a developing solution containing the remaining components of the signal-generating system (the label is also a member of the signal-generating system). When the analyte is present in the test solution, a signal will be generated at the small site. This signal can be distinguished from the signal generated in the part adjacent to the site.

In a variation of the above embodiment, the volume of the test solution is such that it permeates only one part of the strip, that is to say that the liquid reception of the dry part of the strip is at least equal to the liquid reception of the part from the end to the sub-site. Sufficient to penetrate. The end of the strip, which was previously in contact with the test solution, is then contacted with the developer. The color-developing liquid moves in the strip by capillary action and passes through a small portion. At this time, the remaining test solution passes through the small site due to the developing solution. A signal is generated if the analyte is present in the test solution. In another variation of the above embodiment, the first sbp
Do not label ingredients. The assay is performed similarly, but the labeled sbp component complementary to the first sbp component is included in the developer. If the analyte is present, the first sbp component binds to the site, thereby binding the labeled sbp component to the site. In this embodiment, it is often the case that some components of the signal-generating system are not included in the developer and, after contact with the developer, a solution containing the components that were not included (ie, the remaining components) is contacted with the strip. preferable.

In the above aspect, multiple polyvalent analytes can be quantified. For this purpose, several sites are used that are remote from the edges. At one site, the second sbp component that recognizes the determinant site on the first sbp component is non-diffusively bound to the absorbent strip. At the other site, another second sbp component that recognizes the determinant site on the second analyte is non-diffusively bound. Perform the same operation until a small area is formed only for the type of analyte to be tested. The sample to be analyzed is then
Mix with a plurality of labeled sbp components in an appropriate aqueous medium to prepare a test solution. The labeled first sbp component contains an sbp component that recognizes a determinant site on the first analyte, which site binds the first analyte on the absorbent strip to the second sbp component.
It is different from the determinant site recognized by the bp component. Other labeling first sbp
The component is sbp that recognizes the determinant site on the second analyte
It contains a component, which is distinct from the determinant site recognized by the second sbp component that binds the second analyte on the absorbent strip. The number of labeled sbp components corresponds to the number of analytes tested. The presence of one or more analytes to be tested in the sample results in the formation of a complex between each analyte and its corresponding labeled first sbp component. In the absence of analyte, no such complex will form. The test solution penetrates into the strip when the end of the absorbent strip is contacted with the test solution. Both complexes of analyte and labeled sbp contacts bind to each of the above sites on the strip. In the absence of analyte, no complex is formed and therefore the labeled first sbp component corresponding to that analyte does not bind at this site and does not generate a signal. The assay can be designed to use a single developer for all analytes tested. If a particular analyte is present in the sample, a signal will be generated at that particular site. In a particularly preferred variant of this method, the first sbp component is unlabeled and the developer comprises a labeled sbp component complementary to the first sbp component. The strip is contacted with the remaining components of the signal generating system and then with the developer.

In another aspect of the invention, the analyte is a monovalent drug. A sample suspected of containing a drug is mixed with the labeled first sbp component in an appropriate medium to prepare a test aqueous solution. Sign 1s
The bp component is attached to the drug. A second sbp component is supplied to the small portion of the absorbent strip, and the sbp component is labeled the first sbp component.
Recognize determinants on the component (different from the determinants involved in binding the labeled sbp component to the drug). For example, the second sbp
The component may recognize a determinant site on the labeled portion of the labeled first sbp component or on the sbp component portion. In this embodiment, at least all of the labels in the absence of drug in the test sample
A sufficient amount of analyte or drug analog to bind the sbp component must be attached to the strip. Typically, the analyte analog is a derivative of the test drug and binds to the strip in substantial excess, at least between the strip edge and the site. The drug analog is preferably a drug derivative, but other drug analogs, such as antibodies against the idiotypic antibody of the drug, may be used. The sample and the labeled first sbp component are mixed to form a test solution, and when the drug is present in the sample, a complex of the drug and the labeled first sbp component is formed. The complex of drug and labeled first sbp component migrates through the absorbent strip to reach the site and
It binds to that site by binding to the second sbp component, which is specific for the bp component. If the drug is not present in the sample, no complex will form between the drug and the labeled first sbp component. When the test solution is contacted with the end of the absorbent strip, the labeled first sbp component that is not bound to drug binds to the drug analog that is non-diffusively bound to the strip. This drug analog is present in excess so that unbound labeled sbp components do not reach the small site. The test strip is then developed and the generation of a signal at the small site indicates the presence of the drug in the sample.

In the latter embodiment of testing monovalent drugs, multiple drug test solutions may be assayed. In this case, the test solution is prepared by mixing the sample with the labeled first sbp components corresponding to the analytes to be tested in a suitable liquid. If it is desired to test for the presence of any one drug only, the absorbent strip will have the same sites as for the single drug test. However, it is necessary that there be a corresponding drug analog on the strip for each drug tested. If it is necessary to know which drug is present, the strip will be the first sbp corresponding to that drug.
It contains the site to which the second sbp component, which binds specifically to the component-specific determinant site, is attached. The unique determinant site is preferably formed by attaching a hapten to the labeled first sbp component, using an antibody to the hapten as the second sbp component.

For convenience, the device may be supplied as a pre-metered packaged kit of reagents used in the analyte assay. When using an enzyme as a label,
The reagents include the enzyme-labeled first sbp component, a substrate for the enzyme or its precursors (including additional substrates, enzymes and cofactors), and substances that react with the enzymatic products necessary to provide the detectable chromophore or fluorophore. Including. In addition, it may contain other additives such as auxiliary reagents (eg stabilizers, buffers, etc.). The relative amounts of the various reagents can be widely varied to produce reagent concentrations that substantially optimize the sensitivity of the assay. Reagents may be supplied as dry powders (usually lyophilized and contain excipients), such reagents providing a reagent solution at a concentration suitable for conducting the assay when dissolved.

[Examples] Hereinafter, the present invention will be further described with reference to Examples. Before describing the examples, a set of terms is defined.

PBS: Phosphate buffered saline DMF: Dimethylformamide BSA: Bovine serum albumin HCG: Human chorionic gonadotropin HRP: Horseradish peroxidase PO ₄ : Mono- and dibasic sodium phosphate EDTA: Ethylenediaminetetraacetic acid SAMSA: S -Acetylmercaptosuccinic anhydride SMCC: Succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate Phosphate buffer: 10 mM PO ₄ , pH = 7.40 PBS: 10 mM PO ₄ , pH = 7.40 150 mM NaCl buffer A: 10 mM PO ₄ , pH = 7.40 150 mM NaCl 0.1% BSA 0.05% Tween-20 (manufactured by Sigma Chemical Co.) Conjugate buffer: 100 mM PO ₄ , pH = 7.40 150 mM NaCl 0.1% BSA 0.05% Tween-20 developer buffer: 10 mM PO ₄ , pH = 7.00 20 mM NaCl 0.1% BSA 0.005% Triton QS-44 (manufactured by Sigma) 200 μg / ml 4-chloro-1-naphthol 1 mM H ₂ O ₂ Carbonate buffer: 50 mM Na ₂ CO ₃ , pH = 9.50 PO ₄ -NaCl-EDTA buffer: 100 mM Na PO ₄ , pH = 7.50 100 mM NaCl 5 mM EDTA column buffer: 100 mM Na ₃ PO ₄ , pH = 7.00 200 mM NaCl 0.02% NaN ₃ Performed Example 1 Preparation of conjugate of HRP and HCG antibody Preparation of HRP-SAMSA 60 mg of HRP (type IV, manufactured by Sigma) was dissolved in 2 ml of carbonate buffer and dialyzed to remove contaminants.

SAMSA (manufactured by Sigma) was set to 100 mM in dry DMF. HRP
30 mg was reacted with a 12-fold molar excess of SAMSA for 1 hour. PO ₄ -
Sephad equilibrated with NACl-EDTA buffer
The product was purified from free SAMSA using an ex) G-25 small column.

To obtain free-SH groups from SAMSA, the product was incubated with 1/10 volume of 1M NH ₂ OH in PBS for 1 h with stirring in PBS. The resulting product was purified on another Sephadex G-25 column and used immediately thereafter.

Preparation of antibody-SMCC Kohler et al., Nature (1975) 265
Vol. 495-197, HCG β subunit monoclonal antibodies were prepared.

Antibody (4-5 mg / ml) was dialyzed against PO ₄ -NaCl-EDTA buffer to remove contaminants. After dialysis, 8 mg of antibody was added in 25-fold molar excess of SMCC (Pierce Chemical Co.)
Manufactured by K.K.) and stirred at room temperature for 2 hours. Above SMCC
Was freshly prepared as a 100 mM solution in dry DMF.

The product was purified on a Sephadex G-25 small column and used immediately thereafter. Column was equilibrated with PO ₄ -NaCl-EDTA buffer.

Conjugation of HRP-SAMSA and antibody-SMCC Antibody-SMCC and a 12-fold molar excess of HRP-SAMSA were mixed and reacted at room temperature for 4 hours. The reaction was stopped by adding 1 mM β-mercaptoethanol and then 15 mM afterwards 2 mM N-ethylmaleimide.

Purification Column buffer equilibrated with Sephacry
l) The conjugate was separated from free antibody and HRP by size exclusion chromatography using S-300.

Preparation of antibody-bound beads Polybead-carboxylate Microspheres (3.92μ in diameter)
m, 2.5% suspension, Polysciences (Polysciences, Model 9850) was used. 400 μl (10 mg) of beads were washed with a phosphate buffer and suspended in 5 ml of a 2 mg / ml phosphate buffer solution of a monoclonal antibody prepared according to a conventional method (Koehler et al., Supra). This monoclonal antibody recognizes only the α subunit of HCG.

The beads were incubated in the antibody solution overnight at room temperature with agitation to keep the beads in suspension. The beads were then centrifuged, the antibody solution decanted and the beads washed with 5 ml each of phosphate buffer, PBS and buffer A.

After washing with buffer A, the beads were suspended in PBS + 10 mg / ml BSA (5 ml) (to block non-specific binding) and allowed to stand at room temperature for 1 hour.
Mixed for hours.

The beads were then centrifuged and then resuspended in 400 μl of buffer A.

Test Device Preparation Protein (Polyclonal Antitheophylline) coated Whatman 31ET paper was used. The paper was cut into strips 6 mm wide and 9 or 18 cm long (depending on the assay protocol). The paper strip was placed in a 4.5 cm diameter glass frit filter holder. A vacuum was applied and the strip was moistened with distilled water.

10 μl of antibody-bound bead suspension prepared as described above
The paper (containing 250 μl of beads) was absorbed onto the paper by feeding the strip surface twice with a 5 μl capillary tube. The beads were then washed onto the paper with 1 ml of buffer A. The strip was vacuum dried.

Assay A. HCG solution (buffer A or 20 ng / ml in urine) 100 μl
Was mixed with 100 μl of HRP-HCG antibody conjugate solution (1000-fold diluted with conjugate buffer). 2 mm of the test device (length 9 cm) was dipped into the mixture until the top of the medium reached the top of the test device. Then
The test apparatus was immersed in 9 ml of the developer buffer for 10 minutes and then taken out. The thin strip across the test device, where the antibody-bound beads were present, turned blue gray.

As a control, 100 μl of HCG solution instead of 100 μl of buffer A
The above method was carried out using l. No color band appeared on the test device due to the absence of HCG in the test solution.

B. 100 μl of HCG solution was mixed with HRP-HCG conjugate solution. 2 mm of the test device (18 cm in length) was immersed in the mixture, and the whole mixture was allowed to penetrate the test device (about 10 minutes).
2 mm of the test device was further dipped in 200 μl of the developer buffer and allowed to penetrate the test device for another 20 minutes. The thin strip across the test device, where the antibody-bound beads were present, turned blue gray.

As a control, 100 μl of HCG solution instead of 100 μl of buffer A
The above method was carried out using l. No color band appeared on the test device due to the absence of HCG in the test solution.

Prepare CDI coated Whatman 31ET paper of Example 2 A.THC coated paper, moistened with 0.2% Et ₃ N-THF solution and then 11-nor - △ ⁹ -THC-9- carboxylic acid (THC = tetrahydrocannabinol) When 2,2 ¹ - oxybis 5mg / 100ml0.2% amide of (ethylamine) Et ₃ N-
The solution was immersed in a THF solution overnight in a closed container. Unreacted CDI sites were capped with 0.4% ethanolamine in THF, then the paper was washed thoroughly with methanol to remove unreacted THC. The paper was dried, coated with 50 mg MPBS containing 1 mg / ml ovalbumin, 0.25% bile salt, dried and used in the assay.

B. Conjugation of HRP and anti-THC monoclonal antibody Operation 1: Purification of antibody Kohler et al., Nature (1975) 265
Vol. 495-497, the THC monoclonal antibody was prepared and purified by chromatography using a Protein A column.

Procedure 2: Preparation of maleimidated antibody 1. The antibody was dialyzed overnight at 4 ° C. in 10 mM PO ₄ , 150 mM NaCl, pH = 7.0 (without azide).

2. Prepare a new solution of 0.05M SMCC in dry DMF.

3. The antibody was equilibrated at room temperature.

4. Add SMCC to the antibody in a 25: 1 molar ratio and stir the mixture,
Sealed and incubated at 30 ° C. for 2 hours.

5. Excess SMCC was removed by dialysis.

Operation 3: thiolated HRP (DTP-HRP) to (DTP = dithiopyridyl) 1.HRP, dialyzed overnight at 4 ° C. against _{10mM PO 4 / 150mM NaCl / pH7.5} , were equilibrated at room temperature.

2. Prepare a new ethanol solution of 20 mM SPDP (SPDP
= N-succinimidyl-3- (2-pyridyldithio)
Propionate). SPDP was then added to HRP at a molar ratio of 3.5: 1, mixed briefly with stirring, sealed with parafilm and incubated for 30 minutes at room temperature.

_{3.10mM PO 4 / 150mM NaCl (pH7} ) with Sephacryl equilibrated (Sephacryl) in excess through the sample to the S-300 column SP
The DP was removed and then the sample was dialyzed against PO ₄ / NaCl / pH7 buffer overnight at 4 ° C (stored at 4 ° C).

Procedure 4: Activation of thiols on HRP-DTP 1. Sufficient HRP-DTP was taken, which was at least 15-fold excess of the amount of IgG-Mal produced.

Among _{2.10mM PO 4 / 150mM NaCl / pH7.0} , was prepared a new 1M DTT (dithiothreitol).

3. 25 μl of DTT was added per 1 ml of HRP-DTP to make the final concentration of DTT 25 mM, and the reaction was carried out at room temperature for 20 minutes.

4. The sample is then passed through a Sephadex G-25M column where the bed volume is at least 10 times the sample volume and 10 mM PO ₄ /
Equilibrated at room temperature in 150 mM NaCl / 10 mM EDTA / pH 7.0.

Procedure 5: Condensation of Mal-Ab and HRP-SH 1.8 mg of maleimidated antibody and a 15-fold molar excess of deprotected thiolated HRP were mixed and allowed to react at room temperature for 4 hours. Mal
The excess reactive Mal groups were capped by adding to the reactants a 40-fold molar excess of β-mercaproethanol over -Ab.

Procedure 6: Purification of HRP-Ab conjugate 1. The capped conjugate was concentrated by ultrafiltration.

2. Sepharose 4B column (column bed volume 3
The 100 ml), equilibrated with _{10mM PO 4 / 150mM NaCl / pH7.4} .

3. Passed conjugate through column.

C. Antibody-bound beads Rabbit anti-mouse beads (manufactured by BIORAD) were used.

D. Test Device Rabbit anti-mouse beads were linearly supplied at a position 3 cm in height from the lower end of the THC-coated paper.

E. Assay A 75 ng / ml THC solution was used as a sample.

Assay A of Example 1 was repeated as described in Example 1, except that a test device with HRP-Ab conjugate, THC solution as sample, and rabbit anti-mouse beads was used.

A narrow color band was observed on the bead line, indicating the presence of THC in the sample.

The present invention offers many important advantages over known methods. The main advantage of the present invention is that a single assay can be used to quantify multiple analytes in a single assay. This saves time and money.
Further costs can be saved because reagents and devices can be manufactured easily and inexpensively. If the assay device alone can measure the assay results and the signal generated is color or fluorescence, the device does not need to be assisted by other instruments. That is, a control is provided. Positive results are easily distinguished from the background that appears on the test device as a result of non-specific interactions. Also, background signal-generating factors act substantially similarly to sites and remaining areas on the test device.

Another advantage of the present invention is that it does not require cumbersome column operations. The assay device is an absorbent strip that is easy to handle. It is also an advantage that the analyte concentrates in a small area (ie a small area). In many assays, analytes are present in very small amounts, which is difficult to measure. Focusing the analyte on a small area enhances the accuracy of the measurement, especially when only a small amount of the analyte is present.

Another advantage of the present invention is that one member of the sbp component, typically the labeled sbp component, may be used in excess. Excessive use of the sbp component aids the sbp component complex formation reaction.

As described above, in order to clarify the present invention and understand the present invention,
While the invention has been described in detail by the description and examples, it will be understood that changes and modifications can be made within the scope of the invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Edwin Whitscher Ullman, Atherton, Selby Lane 135, Calif. No. 1416 (56) Reference JP-A-53-47894 (JP, A) JP-A-58-76763 (JP, A) JP-A-59-28662 (JP, A) JP-A-59-102388 (JP, A) )

Claims (15)

[Claims] 1. A method for assaying an analyte in a sample suspected of containing an analyte which is a member of a specific binding pair ("sbp component") comprising a ligand and a receptor complementary thereto, comprising: (a) 1s that can bind to the sample and the analyte
A test solution containing the bp component is contacted with a strip end of an absorbent material capable of penetrating the test solution by capillary migration, the strip being substantially larger than the strip area on a strip remote from said end. Has a second sbp component non-diffusively bound to a small site of a small area, the second sbp component has a property of binding to a complex formed by binding the first sbp component to alanite, Furthermore, when the first sbp component is not bound in the complex with the analyte, it is capable of binding the first sbp component, and the analyte analog capable of binding the first sbp component is at least the small portion and the end. Non-diffusively bound to the strip between (b) allowing the test solution to permeate at least a portion of the strip by capillary migration, and (c) strip the (1) at least one component. Is a label conjugated with the sbp component and contains a component of the signal-generating system necessary to generate a measurable signal in the small site according to the amount of the analyte bound to the small site. By contacting with a liquid, the small portion is contacted with a test solution and then with a developer,
(2) If there is another conventional auxiliary component in the signal generating system, the auxiliary component is further contacted with the auxiliary component, and the signal generating system can be detected at the small site according to the amount of the analyte in the sample by a conventional external means. Different signals, and (d) comparing the detectable signal at the small site with the detectable signal at a portion of the strip other than the small site. 2. A particle conjugated with a second sbp component,
The method of claim 1 wherein the strips are non-diffusively bonded to the small areas. 3. The method according to claim 1 or 2, wherein the small portion is a strip-shaped portion that crosses the direction in which the test solution penetrates into the strip. 4. If the analyte is present in the test solution,
4. A method according to any one of claims 1 to 3 wherein the signal generated at the small site has a distinctive pattern with sharp edges which is in sharp contrast to the signal generated at the proximal site of the strip. 5. The signal generating system contains two kinds of enzymes, and the second enzyme uniformly binds to at least the small portion of the strip and a portion in the vicinity of the small portion, and the second enzyme The method according to any one of claims 1 to 4, wherein the product is a substrate for the other enzyme. 6. A method for quantifying a plurality of analytes in a test solution comprising non-diffusively binding a plurality of second sbp components to a plurality of small sites remote from the ends of the strip, The first sbp component binds to the sample in the test solution and
The method according to any one of claims 1 to 5, wherein the 2 sbp component can specifically bind to the corresponding 1 sbp component, respectively. 7. A method for measuring an analyte in a sample suspected of containing an analyte, comprising: (a) penetrating the test solution containing the sample and the antibody of the analyte and the test solution by capillary migration. A strip of absorbent material that is in contact with the strip, and on a strip distant from said strip, a small site of substantially smaller area than the strip area has a member of the specific binding pair (the "sbp component"). ) Is bound non-diffusively, the sbp component is capable of binding to the complex formed by the antibody and the analyte, and the antibody is not bound in the complex with the analyte. Is capable of binding an antibody, and an analyte analog capable of binding an antibody is non-diffusively bound to the strip between the small site and the end, and (b) the test solution is transferred by capillary migration. At least one component is conjugated to the sbp component, and (c) the label is conjugated to at least one component depending on the amount of analyte bound to the small region. (2) a signal generating system by bringing the small portion into contact with a test solution and then with a developing solution by contacting with a developing solution containing components of a signal generating system necessary for measurable signal generation. If there is another conventional auxiliary component in contact with it, the signal generating system can generate a detectable signal according to the amount of the analyte in the sample by the conventional external means at the small site, And (d) comparing the detectable signal at the small site with the detectable signal at a portion of the strip other than the small site. The method according to any one of items 1 to 6. 8. The method according to claim 7, wherein the antibody is an antibody against human chorionic gonadotropin. 9. A method for quantifying a plurality of analytes in a sample, comprising mixing a mixture of antibodies specific for each analyte with the sample and non-diffusively binding a plurality of particle population sets to a strip. 9. Any of claims 7-8, wherein each set of particles comprises a non-diffusively bound sbp component capable of binding to at least one of the complexes formed from the specific antibody and the analyte. The method described in crab. 10. The method of claim 9 wherein the analyte analog of each analyte is attached to a strip. 11. A specific binding pair (sb) complementary to an analyte.
A device for measuring an analyte in a test solution containing the first sbp component of (p component) and a sample suspected of containing the analyte, which has an end for contacting the test solution and moves the test solution by capillary movement. Absorptive strip that can be penetrated by and a non-diffusive second sbp component different from the analyte that is non-diffusively bound to a small area that is substantially smaller than the strip area, away from the end of the strip, where the second sbp The ingredients are
Having the property of binding to the complex formed by the binding of the first sbp component and the analyte, when the first sbp component is not further bound in the complex with the analyte,
A device capable of binding the first sbp component, wherein the analog of the analyte capable of binding the first sbp component is non-diffusively bound to the strip at least between said subsite and end. 12. The device of claim 11 wherein the particles conjugated with the second sbp component are non-diffusively bound to the site of the strip. 13. An apparatus for measuring a plurality of analytes in a test solution, wherein each set of the second sbp component group is non-diffusively bound to different small sites apart from the end of the strip. 13. The device of claims 11 or 12, wherein each second sbp component is capable of binding to a specific complex of analyte and first sbp component. 14. A specific binding pair (sb) complementary to an analyte.
A kit for measuring an analyte in a test solution containing a sample suspected of containing the first sbp component of p component) and the analyte, comprising: (a) having an end portion for contacting the test solution, and moving the test solution by capillary movement. Absorptive strip that can be penetrated by and a non-diffusive second sbp component different from the analyte that is non-diffusively bound to a small area that is substantially smaller than the strip area, away from the end of the strip, where the second sbp The ingredients are
It has the property of binding to the complex formed by the binding of the first sbp component and the analyte, and further when the first sbp component is not bound in the complex with the analyte,
A device capable of binding a 1 sbp component, wherein the analog of the analyte capable of binding the first sbp component is non-diffusively bound to the strip at least between the small site and the end (b) Analyte Is a label conjugated to the sbp component, at least one of which is necessary to generate a detectable signal in the presence of A kit comprising a packaged combination of signal generating system components necessary for possible signal generation and other conventional auxiliary components, and (c) other optional auxiliary substances. 15. A strip of absorbent material having an end for contacting a test solution, said test material being capable of penetrating the test solution by capillary movement, separated from the end by a small area substantially smaller than the strip area. Applying to the site a pattern in which a second sbp component different from the analyte, non-diffusively bound, is applied as a pattern to allow the second sbp component to be absorbed by the strip, where the size of the particle is It is small enough to allow the material to enter the pores but large enough to prevent diffusion through the pores, the second sbp component is due to the binding of the first sbp component and the analyte in the test solution. If the first sbp component is not bound in the complex with the analyte and has a property of binding to the resulting complex, it is possible to bind the first sbp component and bind the first sbp component. Possible A method of making an apparatus for measuring an analyte, wherein the analogue of the analyte is non-diffusively bound to the strip, at least between the subregion and the end.